Oral direct factor Xa (FXa) inhibitors are emerging anticoagulants that have the potential to simplify dosing schemes and hemostatic monitoring in patients with prothrombotic diseases. No specific antidote to their effects exists, however, and this presents a critical unmet clinical need. We hypothesize that the effects of these inhibitors could be overcome by increasing the effective concentration of the enzyme, FXa. Administration of wild type FXa to achieve this is potentially problematic due to its potential for thrombosis. FX is proteolytically activated by the intrinsic or extrinsic pathways of coagulation from the zymogen FX. After proteolytic cleavage and removal of an activation peptide, a newly unveiled N-terminus becomes free to form a salt bridge with Asp[194] in the core of the protein, leading to a conformational change that results in an active protease. Our group has developed variants of FXa that disrupt this new N-terminus and result in a zymogen- like conformation. These variants can be thermodynamically rescued to the protease conformation by binding to the cofactor FVa. Preliminary in vitro studies suggest that these variants have potential as procoagulants to reverse the effects of direct FXa inhibitors. In this proposal, we will evaluate these zymogen-like variants of FXa in in vitro and in vivo hemostasis assays to more fully determine their potential therapeutic utility. Furthermore, we will expand on preliminary data that suggests that direct FXa inhibitors prolong the plasma half-life of FXa by interfering with endogenous plasma inactivators of FXa. In the first aim of our proposal, we will perform a thorough in vitro characterization of FXa zymogen-like variants as reversal agents for direct FXa inhibitors. This will be achieved through purified kinetic assays, thrombin generation assays, and rotational thromboelastography. Another aspect of this aim will be to use purified kinetic assays and plasma based assays to understand the interplay between direct FXa inhibitors and plasma serine protease inhibitors. In the second aim, we will use well-established in vivo hemostasis assays to determine if FXa zymogen-like variants can restore normal hemostasis in anticoagulated mice without inducing thrombosis. We will perform these studies in hemostatically normal mice as well as in mice heterozygous for the FV-Leiden mutation, to better study how these variants would behave in an important prothrombotic population. Through this work, we will gain a better understanding of the interplay between direct FXa inhibitors, FXa, and endogenous inhibitors of FXa. We will also evaluate a potential solution to an urgent hematologic problem.